种子中特异表达IPT基因的植物双元载体构建及转基因水稻获得

摘要

为研究细胞分裂素在水稻籽粒发育中的作用，构建了p1300-pPG-5α-IPT-Nos植物表达载体，并对水稻进行遗传转化。以水稻品种9311为材料，采用PCR法获得种子中特异表达的PG-5α基因启动子，并将此启动子与p1300相连接，构建p1300-pPG-5α载体，用NcoⅠ和SpeⅠ双酶切pSG516，获得IPT-Nos核酸片段，然后将此核酸片段插入到p1300-pPG-5α中，构建p1300-pPG-5α-IPT-Nos植物表达载体。以水稻品种日本晴愈伤组织为材料，采用农杆菌介导的方法进行遗传转化。成功构建了植物表达载体p1300-pPG-5α-IPT-Nos，获得了阳性率为81．3％转IPT基因群体。获得转基因株系后，将为进一步筛选高效表达株系及观察其籽粒生长发育过程提供基础。%In order to specifically express IPT gene in the endosperm of developing rice seed ,the binary vector p1300-pPG-5α-IPT-Nos was constructed,firstly,the promoter of PG-5αwas obtained by PCR amplification from rice genomic DNA(variety:9311)and p1300-pPG-5αwas constructed by inserting pPG-5αinto p1300,then the IPT-Nos fragment was obtained by cutting pSG 516 with NcoⅠ and SpeⅠ.After IPT-Nos inserted into p1300-pPG-5α, p1300-pPG-5α-IPT-Nos was constructed .pPG-5α-IPT-Nos was transformated into Oryza sativa ( subsp .japonicacv . Nipponbare ) cells by agrobacterium mediated method and regenerative seedlings were obtained .The results of PCR showed that about 81 .3%seedlings were positive .